PROJECT SUMMARY Malfunctioning of mitochondrial apoptosis is implicated in a range of neurodegenerative diseases and cancers, where it leads to premature neuronal death or uncontrolled cell growth. Animal studies of neurodegenerative disorders have shown the benefits of drugs that target early apoptotic events in mitochondria, but many critical aspects specifics of the underlying molecular processes remain enigmatic. It is clear that mitochondrial membranes undergo extensive remodeling, that oxidation of lipids plays a critical role, that redistributed lipid species act as critical pro-apoptotic signals, and that mitochondrial proteins attain new apoptosis-specific functions. The latter notably includes mitochondrial cytochrome c (cyt-c), as it performs two essential functions: it catalyzes the oxidation of the mitochondrial lipid cardiolipin by reactive oxygen species, and cyt-c release is a final irrevocable ?death signal?. Informed by extensive preliminary studies, we have defined a new, integrated model that ties together these events, and identifies a potential critical positive feedback loop that underlies the earliest stages of mitochondrial apoptosis. We propose experiments that will test this model, which features at its core a synergy of protein-lipid interactions and membrane structural changes. We will leverage cutting-edge solid-state NMR spectroscopy and complementary techniques to elucidate how cyt-c and cardiolipin combine and interact to trigger the pro-apoptotic peroxidase activity of cyt-c, through the induction of structural changes in the protein. A recent publication and a broad array of preliminary results show how we can use advanced solid-state NMR to provide the residue- and site-specific resolution that will be necessary to truly understand the molecular features of this cardiolipin/cyt-c complex. Additional structural and functional measurements will involve peroxidase assays, fluorescence measurements, electron microscopy, and various optical spectroscopies. We will also detail how polyunsaturated cardiolipin, lipid oxidation, cyt-c, and other apoptotic players impact the structure, fluidity, and stability of mitochondrial membranes. Again solid-state NMR will be a powerful tool that provides unique insights into the molecular structure and dynamics of complex biological membranes. The proposed integrated experimental approach, specifically including the use of state-of-the-art magic-angle-spinning solid-state NMR, will be both essential and also unprecedented for this system. An array of structural, biophysical, and functional measurements will be enabled by the abovementioned complementary experimental techniques and executed by an experienced interdisciplinary research team. Our team has a published track record of collaborative work and brings to bear an in-depth expertise in the key experimental techniques, membrane biophysics, as well as the study of the roles of cyt-c and cardiolipin in apoptosis. Thus, this project is certain to provide much-needed and unprecedented molecular insight into the pivotal early stages of intrinsic apoptosis, and help inform ongoing and future efforts to target these events for drug design, spanning applications from neurodegenerative disease to cancer.